Treatment of autoimmune disease

ABSTRACT

The present invention features a novel combination therapy useful in the treatment of autoimmune disease that increases or maintains the number of functional cells of a predetermined type in a mammal by killing or inactivating autoimmune cells and re-educating the host immune system.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of Ser. No.09/521,064, filed on Mar. 8, 2000, which, in turn, claims benefit ofU.S. provisional application Serial No. 60/123,738, filed on Mar. 10,1999, both of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] Early onset diabetes mellitus, or Type I diabetes, is a severe,childhood, autoimmune disease, characterized by insulin deficiency thatprevents normal regulation of blood glucose levels. Insulin is a peptidehormone produced by the β cells within the islets of Langerhans of thepancreas. Insulin promotes glucose utilization, which is important forprotein synthesis as well as for the formation and storage of neutrallipids. Glucose is also the primary source of energy for brain andmuscle tissue. Type I diabetes is caused by an autoimmune reaction thatresults in complete destruction of the β cells of the pancreas, whicheliminates insulin production and eventually results in hyperglycemiaand ketoacidosis.

[0003] Insulin injection therapy has been useful in preventing severehyperglycemia and ketoacidosis, but fails to completely normalize bloodglucose levels. Although insulin injection therapy has been quitesuccessful, it does not prevent the premature vascular deteriorationthat is the leading cause of morbidity among diabetics today.Diabetes-related vascular deterioration, which includes bothmicrovascular deterioration and acceleration of atherosclerosis, caneventually cause renal failure, retinal deterioration, angina pectoris,myocardial infarction, peripheral neuropathy, and atherosclerosis.

[0004] A promising treatment for diabetes, islet transplantation, hasbeen in human clinical trials for over ten years. Unfortunately, theresults where Type I diabetes is the underlying etiology are poor. Therehave been many successes with islet transplantation in animals, but onlywhere the animals are diabetic due to chemical treatment, rather thannatural disease. The only substantiated peer reviewed studies usingnon-barrier and non-toxic methods and showing success with islettransplants in naturally diabetic mice use isogeneic (self) islets. Theisogenic islets were transplanted into already diabetic NOD micepre-treated with TNF-alpha (tumor necrosis factor-alpha); BCG (bacillusCalmette-Guerin, an attenuated strain of mycobacterium bovis); and CFA(complete Freund's adjuvant), which is an inducer of TNF-alpha(Rabinovitch et al., J. Immunol. (1997)159(12):6298-303). This approachis not clinically applicable primarily because syngeneic islets are notavailable. In the allograft setting of islet transplantation, the graftsare rejected presumably due to autoimmunity. Furthermore, diabetic hosttreatments such as body irradiation and bone marrow transplantation aretoo toxic in Type I diabetes patients, rendering the short-termalternative of insulin therapy more attractive.

[0005] I previously developed a transplant method to introduceallogeneic and xenogeneic tissues into non-immunosuppressed hosts, inwhich the cells are modified such that the donor antigens are disguisedfrom the host's immune system (Faustman U.S. Pat. No. 5,283,058, herebyincorporated by reference). Generally, masked islets or transgenicislets with ablated class I are only partially protected from recurrentautoimmunity in spontaneous non-obese diabetic (NOD) mice (Markmann etal., Transplantation (1992) 54(6):1085-9). There exists the need for atreatment for diabetes and other autoimmune diseases that halts theautoimmune process.

SUMMARY OF THE INVENTION

[0006] The present invention provides a novel method for reversingexisting autoimmunity.

[0007] Accordingly, the invention provides a method for increasing ormaintaining the number of functional cells of a predetermined type(e.g., islet cells) in a mammal, involving the steps of: (a) providing asample of cells of the predetermined type, (b) treating the cells tomodify the presentation of an antigen of the cells that is capable ofcausing an in vivo autoimmune cell-mediated rejection response, (c)introducing the treated cells into the mammal, and (d) prior to, after,or concurrently with step (c) treating the mammal to kill or inactivateautoimmune cells of the mammal.

[0008] In preferred embodiments, step (b) involves eliminating,reducing, or masking the antigen, which is preferably is MHC class I.Such methods are known, and are described, e.g., in Faustman, U.S. Pat.No. 5,283,058.

[0009] Preferably, step (d) involves administering to the mammal tumornecrosis factor-alpha (“TNF-alpha”), or a TNF-alpha inducing substance,(i.e., an agonist). As will be explained in more detail below, theTNF-alpha signaling pathway is an inflammatory pathway that effectivelybrings about killing of the autoimmune cells that attack the desiredcells. There are many methods for stimulating TNF-alpha production,including the following: vaccination with killed bacteria or toxoids,e.g., BCG, cholera toxoid, or diphtheria toxoid; induction of limitedviral infections; administration of LPS, interleukin-1, or UV light;activation of TNF-alpha producing cells such as macrophages,B-lymphocytes and some subsets of T-lymphocytes; or administration ofthe chemotatic peptide fMET-Leu-Phe; CFA-pacellus toxoid, Mycobateriumbovis bacillus, TACE (a metalloproteinase that mediates cellularTNF-alpha release), hydrozamates, p38 mitogen activated protein (“MAP”)kinase, and viral antigens that activate NF_(κ)B transcription factorsthat normally protect the cells from apoptosis (i.e., cell death).

[0010] Killing of undesired autoimmune cells can also be accomplished byadministering agents that act as agonists for the enzyme, TNF-alphaconverting enzyme, that cleaves the TNF-alpha precursor to producebiologically active TNF-alpha.

[0011] Autoimmune cells can also be killed by administering agents thatdisrupt the pathways that normally protect autoimmune cells from celldeath, including soluble forms of antigen receptors such as CD28 onautoreactive T cells, CD40 on B cells that are involved in protection ofautoimmune cells, and CD95 (i.e., Fas) on T-lymphocytes. Other suchagents include p75NTF and lymphotoxin Beta receptor (LtbetaR).

[0012] The methods of the invention in some respects run counter tocurrent treatment regimens for autoimmune diseases. Many of the majorapproved therapies for such diseases involve the administration ofanti-inflammatory drugs that inhibit the production of TNF-alpha,including COX-2 inhibitors, and TNF antagonists. My studies indicatethat these conventional therapies are actually deleterious, in that theybring about expansion of the population of harmful autoimmune cells inthe patient, increasing the number and severity of lesions andautoreactive infiltrates. In addition, many of these anti-autoimmuneinflammatory drug therapies cause severe re-bound disease afterdiscontinuation. For example, treatment with anti-inflammatory agentsactually increases the number of lymphocyte infiltrates in the pancreasof a diabetic. Once treatment is discontinued, these lymphocytes regaintheir normal function, resulting in a heightened autoimmune response.

[0013] The methods of the invention can be used to treat any of themajor HLA class II-linked autoimmune diseases characterized bydisruption in MHC class I peptide presentation and TNF-alphasensitivity. These diseases include, for example, type I diabetes,rheumatoid arthritis, SLE, and multiple scelorosis. The method can beused in any mammal, e.g., human patients, who have early pre-symptomaticsigns of disease, or who have established autoimmunity.

[0014] The invention also provides a method for increasing ormaintaining the number of a predetermined type e.g., islet cells, in amammal by the steps of (a) treating the mammal with an agent that killsor inactivates autoimmune cells of the mammal; (b) periodicallymonitoring the cell death rate of the autoimmune cells; and (c)periodically adjusting the dosage of the agent based on the informationobtained in monitoring step (b).

[0015] In any of the methods of the invention in which TNF-alpha isadministered or stimulated, two agents can be used together for thatpurpose, e.g., TNF-alpha and IL-1 can be used in combination therapy, ascan any other combinations of agents.

[0016] In addition, the invention provides a method for diagnosing anautoimmune disease or predisposition to such a disease in a mammal(e.g., a human patient). The method includes the steps of (a) providingperipheral cells from a mammal; (b) treating these cells with aTNF-alpha treatment regimen and; (c) detecting cell death in theperipheral cells, where an increase in cell death, when compared withcontrol cells, is taken as an indication that the mammal has anautoimmune disease or predisposition to such disease. This diagnosticmethod can be used for any mammal, however, the preferred mammal is ahuman patient. Peripheral cells that can be used in this method are, forexample, splenocytes, T lymphocytes, B lymphocytes, or cells of bonemarrow origin and in step (b) of the method, these cells are preferablytreated with TNF-alpha.

[0017] By “functional cell,” is meant cells that carry out their normalin vivo activity. In certain preferred embodiments of the invention, itis preferred that the cells are capable of expressing endogenous selfpeptide in the context of MHC class I.

[0018] By “predetermined type,” when used in reference to functionalcells, is meant that one may select a specific cell type. For example,one skilled in the art may decide to carry out the method of the presentinvention in order to increase or maintain the number of functionalislet cells in the pancreas. In this example, the predetermined celltype is islet cells.

[0019] By “class I and peptide” is meant MHC class I presentation ofpeptide (i.e., self peptide) on the cell surface. Cytoplasmic antigensare believed to be processed into peptides by cytoplasmic proteases andat least in part by the proteasome, a multicatalytic proteinase complexof which the Lmp2 protein, discussed herein, is associated. The processof MHC class I presentation is thought to include formation of a complexbetween the newly synthesized MHC class I molecule, including aglycosylated heavy chain non-covalently associated withβ2-microglobulin, and peptide within the rough endoplasmic reticulum ofthe cell. Thus, “class I and peptide” refers to the MHC class I/peptidecomplex as it is presented on the cell surface for education of theimmune system.

[0020] By “killing” or “kills” is meant to cause cell death byapoptosis. Apoptosis can be mediated by any cell death pathway.According to the present invention, cells that are susceptible tokilling are defective in protection from apoptosis due to a defect in acell death pathway.

[0021] “Autoimmune cells,” as used herein, includes cells that aredefective in protection from apoptosis. This defect in protection fromapoptosis can be in the pathway linked to TNF-induced apoptosis, or anapoptotic pathway unrelated to TNF. Autoimmune cells of the presentinvention include, for example, adult splenocytes, T lymphocytes, Blymphocytes, and cells of bone marrow origin, such as defective antigenpresenting cells of a mammal.

[0022] By “defective” or “defect” is meant a defect in protection fromapoptosis.

[0023] By “exposure” is meant exposure of a mammal to MHC class I andpeptide (i.e., self peptide or endogenous peptide) by any means known inthe art. In one preferred embodiment, exposure to MHC class I andpeptide is carried out by administering to the mammal an MHC classI/peptide complex. In other preferred embodiments, exposure to MHC classI and peptide occurs by exposing the mammal to cells that express MHCclass I and peptide.

[0024] By “cells capable of expressing MHC class I and peptide” ismeant, for example, cells that are class I⁺ or cells that are classI^(−/−) (e.g., cells having a mutation in the β₂M gene) but that arereconstituted in vivo by a compensatory component (e.g., serum β₂M).

[0025] By “maintenance of normal blood glucose levels” is meant that amammal is treated, for example, by insulin injection or by implantationof a euglycemic clamp in vivo, depending on the host being treated.

[0026] By “lmp2 gene or an equivalent thereof,” is meant a cell that hasa defect in prevention of apoptotic cell death, for example, a cell thathas an ablation at a critical point in an apoptotic cell death pathway.In another aspect, “lmp2 gene or an equivalent thereof” means that acell has a mutation in the lmp2 gene or a gene that carries out afunction the same as or similar to the lmp2 gene (i.e., a gene encodinga proteasome subunit). Alternatively, the phrase “lmp2 gene or anequivalent thereof” can be used to refer to a cell that has a mutationin a gene that encodes a regulator of the lmp2 gene or another componentof the proteasome complex. For example, a human homolog of the murinelmp2 gene is an equivalent of the lmp2 gene according to the presentinvention. As but another example, a gene that carries out the same orsimilar function as the lmp2 gene, but that has a low amino acidsequence similarity, would also be considered as an equivalent of thelmp2 gene according to the present invention.

[0027] “Combination therapy,” or “combined therapy,” as used herein,refers to the two-part treatment for increasing the number of functionalcells of a predetermined type that includes both (1) ablation ofautoimmune cells, and (2) re-education of the host immune system.

[0028] By “TNF-alpha induction,” “TNF-alpha treatment regimen,” or“TNF-alpha” includes the administration of TNF-alpha, agents that induceTNF-alpha expression or activity, TNF-alpha agonists, agents thatstimulate TNF-alpha signaling, or agents that act on pathways that causeaccelerated cell death of autoimmune cells, according to the invention.Stimulation of TNF-alpha induction (e.g., by administration of CFA) ispreferably carried out prior to, after, or during administration (viaimplantation or injection) of cells in vivo.

[0029] By “effective,” is meant that the dose of TNF-alpha, or TNF-alphainducing agent, administered, increases or maintains the number offunctional cells of a predetermined type in an autoimmune individual,while minimizing the toxic effects of TNF-alpha administration.Typically, an effective dose is a reduced dose, compared to dosespreviously shown to be ineffective at treating autoimmune disease,particularly established autoimmune disease.

[0030] The methods of the invention provide, for the first time,effective reversal of naturally-occurring (as opposed to chemicallyinduced) mediated diseases such as type I diabetes.

[0031] Other features and advantages of the invention will be apparentfrom the following description of the preferred embodiment thereof, andfrom the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1 shows three graphs that depict blood glucose concentrationat indicated times after transplantation (left panels) and sixphotographs showing the histology of the pancreas (middle panels) andgraft site under the kidney capsule (right panels) of diabetic NODfemale mice subjected to transplantation with islets from various donortypes and a single injection of CFA. Islet grafts were derived fromyoung NOD mice (panel A), C57 mice (panel B), or β₂M^(−/−) C57 mice(panel C).

[0033]FIG. 2 is a graph depicting the histological characteristics ofthe graft site and pancreas of individual NOD hosts subjected totransplantation of islets from various types of donors in the absence orpresence of TNF-alpha induction. Open squares indicate lack of visibleislet structures and of visible lymphocytic accumulation; open squareswith dots indicate massive lymphocytic accumulation obscuring isletremnants; shaded squares indicate viable islets without lymphocytes;shaded squares with dots indicate viable islet structures with onlycircumferential lymphocytic accumulation; panc indicates pancreas.

[0034]FIG. 3 shows five graphs depicting blood glucose levels (leftpanels) and five photographs showing the histology of the pancreas(right panels) of diabetic NOD female mice subjected to transplantationwith islets from β₂M^(−/−) C57 mice and a single injection of CFA.Arrows indicate the time of removal of the kidney containing the isletgraft by nephrectomy.

[0035]FIG. 4 shows two graphs (panels A and B) and three photographs(panels C, D, and E) that demonstrate the effect of TNF-alpha inductionand repeated exposure to C57 splenocytes on islet regeneration andrestoration of normoglycemia in diabetic NOD hosts. Panel A representsNOD females treated with daily injections of insulin alone (controls,n=5). Panel B represents NOD females treated with insulin (untilnormoglycemia was restored) plus a single injection of CFA and biweeklyinjections of 9×10⁶ C57 splenocytes (n=9). Arrows represent time ofdeath. Pancreatic histology of a control animal (panel C); an animalthat remained hyperglycemic (panel D); and an animal in whichnormoglycemia was restored (panel E).

[0036]FIG. 5 shows four graphs (left panel) that depict the effect ofmaintenance of normoglycemia during TNF-alpha induction and splenocytetreatment on islet regeneration in diabetic NOD mice. The graphs areaccompanied by eight photographs that show the histology of thepancreas, specifically islets and associated lymphocytic infiltrates(middle panels) and islet insulin content (right panels). Arrowsrepresent time of removal of euglycemic clamp. Mice received a singleinjection of CFA only (panel A), CFA plus biweekly injections ofsplenocytes (9×10⁶) from normal C57 mice (panel B), β₂M^(−/−),TAP1^(−/−) C57 mice (panel C), or MHC class II^(−/−) C57 mice (panel D).

[0037]FIG. 6 shows six graphs depicting flow cytometric analysis of theeffect of islet regeneration on the percentage of CD3⁺ T cells amongsplenocytes of NOD mice. Percentage CD3⁺ cells is shown in the upperright corner of each graph. Panel A represents a 6- to 7-month-oldfemale C57 mouse; panel B represents a diabetic NOD female treated withinsulin alone for 12 days; panels C through F represent diabetic NODfemales implanted with a euglycemic clamp for ˜40 days and treated witha single injection of CFA either alone (panel D) or together withbiweekly injections of normal C57 splenocytes (panel C), MHC classII^(−/−) C57 splenocytes (panel E), or β₂M^(−/−), TAP1^(−/−) C57splenocytes (panel F).

[0038]FIG. 7 shows the effects of TNF-alpha on the survival of spleencells derived from untreated C57 (C57BL/6) and untreated or treated NODmice. Spleen cells were incubated without or with TNF-alpha (20 ng/ml)for 24 hours after which apoptotic cells were detected by flow cytometrywith fluorescein isothiocyanate-conjugated annexin V. The percentage ofapoptotic cells is given in the top right-hand comer of each panel.

[0039]FIG. 8 shows the blood sugar levels in adoptive transferrecipients of fresh untreated spleen cells (top) or in vitro TNF-alphatreated cells (bottom) from diabetic NOD mice transferred to youngirradiated male NOD mice. In the top panel, mice were injected with2×10⁷ spleen cells pooled from three spontaneously diabetic NOD mice. Inthe bottom panel, recipient mice were also injected with spleen cellspooled from three,spontaneously diabetic NOD mice. Prior to injection,these splenocytes were treated with 10 ng/ml TNF-alpha in vitro andassayed for cell viability. 2×10⁷ viable spleen cells were then injectedinto recipient mice.

DETAILED DESCRIPTION

[0040] The present invention provides a method of increasing ormaintaining the number of functional cells of a predetermined type in amammal by preventing cell death. In preferred embodiments, this methodis used to treat an autoimmune disease where endogenous cell and/ortissue regeneration is desired. Such autoimmune diseases include,without limitation, diabetes melitus, multiple sclerosis, prematureovarian failure, scleroderm, Sjogren's disease, lupus, vilelego,alopecia (baldness), polyglandular failure, Grave's disease,hypothyroidism, polymyosititis, pempligus, Chron's disease, colititis,autoimmune hepatitis, hypopituitarism, myocardititis, Addison's disease,autoimmune skin diseases, uveititis, pernicious anemia,hypoparathyroidism, and rheumatoid arthritis. One aspect of theinvention provides a novel two-part therapeutic approach to ablateexisting autoimmunity while re-educating the immune system via MHC classI and peptide. A key feature of the invention is the discovery thatreexpression of endogenous antigens in the context of class I MHC isessential to terminate an ongoing autoimmune response.

[0041] As mentioned above, Type I diabetes results from destruction ofthe cells of the Islet of Langerhans of the pancreas via a severeautoimmune process. The goal for treatment of Type I diabetic patientsis to permanently halt the gaff autoimmune process so that pancreaticislets are preserved. Alternatively, in cases where islet destructionfrom autoimmunity is complete, the goal is to provide a method ofreplacing islet cells, or allowing them to regenerate. Thus, theinvention provides a novel method for increasing or maintaining thenumber of functional cells of a predetermined type for treatment ofestablished cases of diabetes melitus, where existing autoimmunity isreversed.

[0042] In adult onset diabetes, or Type II diabetes, the β islet cellsof the pancreas are often defective in secretion of insulin. However,recent studies indicate that, in some patients, autoimmune destructionof β islet cells does play an important role in disease progression(Willis et al., Diabetes Res. Clin. Pract. (1998) 42(1):49-53). Thus,the present invention may also be used to treat Type II diabetes wherean autoimmune component is present.

[0043] Relating the Present Invention to Known Genetic and FunctionalInformation

[0044] Genetic and functional studies have identified mutations in thelmp2 gene in NOD diabetic mice, a murine model for human type I diabetes(Li et al., Proc. Natl. Acad. Sci., USA (1994) 91:11128-32; Yan et al.,J. Immunol. (1997) 159:3068-80; Fu et al., Annals of the New YorkAcademy of sciences (1998) 842:138-55; Hayashi et al., Molec. Cell.Biol. (1999) 19:8646-59). Lmp 2 is an essential subunit of theproteasome, a multi-subunit particle responsible for processing a largenumber of intracellular proteins. The pronounced proteasome defect inLmp2 results in defective production and activation of the transcriptionfactor NF_(κ)B through impaired proteolytic processing of NF_(κ)B togenerate NF_(κ)B subunits p50 and p52 and impaired degradation of theNF_(κ)B inhibitory protein, I_(κ)B. NF_(κ)B plays an important role inimmune and inflammatory responses as well as in preventing apoptosisinduced by tumor necrosis factor alpha (TNF-alpha). Autoreactivelymphoid cells expressing the lmp2 defect are selectively eliminated bytreatment with TNF-alpha, or any TNF-alpha inducing agent, such ascomplete Freund's adjuvant (CFA), or an agent that acts on a pathwayrequired for cell death protection, for example, any pathway convergingon the defective apoptotic activation mechanism. This is wellillustrated by faulty apoptosis protection in the NOD mouse which lacksformation of protective NF_(κ)B complexes.

[0045] The lmp2 gene is genetically linked to the MHC locus (Hayashi etal., supra). Antigen presenting cells of NOD mice cease production ofLMP2 protein at approximately 5-6 weeks, a process that terminates theproper processing of endogenous peptides for display in the context ofMHC class I on the cell surface. Surface display of endogenous peptidein the context of MHC class I molecules is essential for the selectiveelimination of T cells reactive to self antigens (Faustman et al.,Science (1991) 254:1756-61; Ashton-Rickardt et al., Cell (1993)73:1041-9; Aldrich et al., Proc. Natl. Acad. Sci. USA (1994)91(14):6525-8; Glas et al., J. Exp. Med. (1994) 179:661-72). Currenttheory suggests that interruption of endogenous peptide presentation viaMHC class I prevents proper T cell education and is responsible for adiverse array of autoimmune diseases (Faustman et al., supra; Fu et al.,J. Clin. Invest. (1993) 91:2301-7). These data are also consistent withthe clear sex-, tissue-, and age-specific differences in the expressionof this error which parallel the initiation and disease course ofinsulin-dependent (type I) diabetes. It is hypothesized that the triggerfor the initiation of autotimmunity is the tissue- anddevelopmental-specific dysregulation of the proteasome (or MHC class I)in islet cells, as opposed to lymphocytes. As mentioned above, it ispossible that this defect triggers a pathologic T cell response to isletcells via interruption of proper T cell education (Hayashi et al.,supra).

[0046] In a normal, non-diabetic, human or animal, peripheral tissues,including islets, consistently express endogenous antigens in thecontext of MHC class I (Hayashi et al., supra). Constitutivetissue-specific display of self peptide via MHC class I could maintainperipheral tolerance in the context of properly selected lymphocytes(Vidal-Puig et al., Transplant (1994) 26:3314-6; Markiewicz et al.Proceedings of the National Academy of Sciences of the United States ofAmerica (1998) 95(6):3065-70). In the absence of such tissue-specificdisplay, poor negative selection of T-lymphocytes could lead tooverexpansion of self-reactive lymphocytes, a prominent feature in humanand murine disease models.

[0047] As mentioned above, autoreactive lymphoid cells expressing thelmp2 defect are selectively eliminated, for example, by treatment withTNF-alpha, or any TNF-alpha inducing agent, such as complete Freund'sadjuvant (CFA). Although the specific gene defect has not beenidentified in human autoimmune patients, it is known that humansplenocytes in the human diabetic patient, like murine splenocytes inthe NOD mouse, have defects in resistance to TNF-alpha induced apoptosis(Hayashi et al., supra). Specific cells in human autoimmune patientsmight express a genetic defect, similar to the proteasome defect inmice, that increases susceptibility to TNF-alpha induced apoptosis or ananalogous apoptotic cell death pathway. Therefore, in patientsexpressing the genetic defect, only the autoimmune cells are killed.Permanently eliminating the autoreactive cells is a key feature of aneffective treatment for an autoimmune disease.

[0048] According to the present non-limiting theory, of the invention,multiple cell death pathways exist in a cell and any one or more ofthese cell death-related pathways may be defective, accentuating thesensitivity of these cells to cell death. For example, susceptibility toTNF-alpha induced apoptosis could occur via a failed cell deathinhibition pathway (e.g., by defective production and activation of thetranscription factor NF_(κ)B, as in the NOD mouse). Further, it is wellknown that there are two different TNF-alpha receptors. Defectivesignaling through either receptor could render autoimmune cellssusceptible to TNF-alpha induced apoptosis. As but another example,defective cell signaling through surface receptors that stimulatepathways that interact with the cell death pathway, i.e., LPS, IL-1,TPA, UV light etc., could render autoimmune cells susceptible toapoptosis according to the theory of the present invention. Therefore,methods of the present invention that are beneficial in the treatment ofautoimmune disease are applicable to any autoimmune patient that has adefect in a cell death pathway.

[0049] As mentioned above, current therapies for autoimmune disease aredirected toward decreasing the inflammatory reaction that is thought tobe responsible for destruction of self. TNF-alpha is part of theinflammatory response. Thus, according to the present theory, inductionof an inflammatory response, rather than inhibition of an inflammatoryresponse, is the preferred method of treating an autoimmune individual.This theory runs counter to existing dogma surrounding autoimmunetherapy today.

[0050] It is possible that TNF-alpha is inducing a cytokine, toxoid, orother related molecule induced in the inflammatory response that is theresponsible for the benefit of TNF-alpha treatment. If so, induction ofinflammation via TNF-alpha treatment is still in agreement with thetheory of the invention. In a preferred embodiment, induction ofinflammation via TNF-alpha treatment induces mediators of autoimmunecell death.

[0051] A Novel Assay for Monitoring Treatment

[0052] It is well known that prolonged TNF-alpha treatment by itself ishighly toxic. In light of the elucidation of the cell death pathwaydescribed above, we hypothesized that the knowledge of this pathwaycould enable development of a sensitive in vitro assay that could beused to monitor the in vivo effect of a particular TNF-alpha treatmentregimen (i.e., any treatment regimen that results in induction ofTNF-alpha and inflammation). More particularly, a monitoring systemcould be developed that combined the administration of TNF-alpha alonewith an assay capable of measuring the effect of TNF-alpha treatment onapoptosis of autoimmune cells in a mammal diagnosed with an autoimmunedisease. Such a monitoring system would make it possible to measure theeffect of particular doses of TNF-alpha on the apoptosis of autoimmunecells concurrently with treatment of an autoimmune individual. Moreover,such a monitoring system would enable optimization or adjustment of thedose of TNF-alpha (i.e., or TNF-alpha inducing agent) to maximizeautoimmune cell death, while minimizing exposure of the mammal to toxicdoses of TNF-alpha.

[0053] Thus, the invention provides a method of increasing ormaintaining the number of functional cells of a predetermined type in amammal that involves a) treating a mammal to kill or inactivateautoimmune cells of the mammal; b) periodically monitoring the celldeath rate of the autoimmune cells (i.e., by assaying the cell deathrate of autoimmune cells in the mammal, wherein an increase in celldeath rate of auto reactive T-lymphocytes indicates an increase in thenumber of functional cells of the predetermined type (i.e., resistant tocell death)); and (c) periodically adjusting the dosage of the agentbased on the information obtained in step (b). The autoimmune cells ofthe present invention include any cell defective in protection fromapoptotic cell death by any stimulus, for example, TNF-alpha, CD40,CD40L, CD28, IL1, Fas, FasL, etc.

[0054] The assay of step (b) allows one to identify novel formulationsof TNF-alpha, TNF-alpha inducing agents, TNF-alpha agonists, or agentsthat act on the TNF-alpha signaling pathway effective in inducingapoptosis of T-lymphocytes or antigen presenting cells, that can beadministered over a longer course of treatment than was possible priorto the present invention (e.g., preferably over a period of months, morepreferably over a period of years, most preferably over a lifetime).

[0055] In a related embodiment, the present monitoring system may beused to identify new doses, durations of treatment, and treatmentregimens for TNF-inducing agents that were previously discounted asuseful treatments because there was no way to monitor their effect. Forexample, in contrast to a preliminary report identifying BCG, aTNF-alpha inducing agent, as a useful type I diabetes treatment(Shehadeh et al., Lancet, (1994) 343:706), researchers failed toidentify a therapeutic dose of BCG because there was no way to monitorthe effect of BCG in vivo (Allen et al., Diabetes Care (1999) 22:1703;Graves et al., Diabetes Care (1999) 22:1694).

[0056] The assay of step (b) may also be used to tailor TNF-alphainduction therapy to the needs of a particular individual. For example,as mentioned above, in one preferred embodiment, the assay of step (b)can be carried out every day or every other day in order to measure theeffect of TNF-induction therapy and/or cell death inducing agents onautoimmune cell death rate so that adjustment to the administered dose,duration of treatment (i.e., the period of time over which the patientwill receive the treatment), or treatment regimen (i.e., how many timesthe treatment will be administered to the patient) of TNF-alpha can bemade to optimize the effect of TNF-alpha treatment and minimize theexposure of the patient to TNF-alpha or other cell death inducingagents. Of course, the skilled artisan will appreciate that the assaycan be performed at any time deemed necessary to assess the effect of aparticular regimen of TNF-alpha induction therapy on a particularindividual (i.e., during remission of disease or in a pre-autoimmuneindividual).

[0057] The assay can be used to tailor a particular TNF-alpha inductionregimen to any given autoimmune disease. For example, the in vitromonitoring of selective killing of autoimmune cells can be used toselectively grade the drug (i.e., adjust the dose administered tomaximize the therapeutic effect). The monitoring system described hereincan be used to monitor in vivo trials of TNF-alpha treatment bycontinuously measuring the elimination of autoimmune cells, e.g.,autoreactive T lymphocytes, with continuing sensitivity. Of course, theskilled artisan will appreciate that the present monitoring system canbe used to measure the effect of TNF-alpha on in vivo killing ofautoimmune cells in cases where TNF-alpha-induction therapy is cited inconjunction with any other therapy, e.g., T cell re-education, asdescribed herein.

[0058] It is well known that TNF-alpha induction therapy has been shownto be ineffective in patients with established autoimmunity, e.g.,established diabetes, but is effective in patients in a pre-autoimmunestate, e.g., patients in a pre-diabetic or pre-lupus state. In addition,it has been established that TNF-alpha induction in adult NOD and NZBmice (a murine strain susceptible to lupus-like disease) decreasesdiabetic or lupus symptoms respectively. According to the invention,TNF-alpha therapy can be effective even in patients with establisheddisease, by monitoring the elimination of autoimmune cells andoptimizing the dose, duration of treatment, and/or re-treatment scheduleaccordingly. Thus, the assay of step (b) may be used to identify aneffective dose, duration of treatment, or treatment regimen of TNF-alpha(e.g., lower than doses previously shown to be ineffective in treatmentof diabetes, particularly in the treatment of established diabetes) thatcan be used as an effective treatment for autoimmune disease.

[0059] In another preferred embodiment, the assay of step (b) is used toidentify a dose, duration of treatment, or treatment regimen ofTNF-alpha that can reduce or eliminate side effects associated with aparticular autoimmune disease. A particular dose of TNF-alpha may beidentified that reduces or eliminates the symptoms associated with, forexample, vascular collapse associated with diabetes, blindness or kidneyfailure associated with Type 1 diabetes, or skin eruptions associatedwith lupus. It is well established that it is the side effectsassociated with the autoimmune reaction that are often responsible formortality of autoimmune patients. Thus, in one preferred embodiment, themonitoring system of the present invention identifies a treatmentregimen for TNF-alpha that reduces the symptoms and/or complications ofthe autoimmune disease, such that the quality of life of the patient isimproved and/or the life-span of the patient being treated is prolonged.In a related embodiment, the monitoring system of the present inventionidentifies a treatment regimen for TNF-alpha that prevents diseaseprogression or even halts disease in a patient diagnosed with anautoimmune disease.

[0060] Thus, in another aspect, the present invention provides amonitoring system for measuring the rate of cell death in an autoimmunemammal, including (a) a treatment regimen for killing or inactivatingautoimmune cells in a mammal; and (b) an assay capable of measuring theeffect of the treatment regimen on the cell death rate of autoimmunecells in the mammal, wherein an increase in cell death rate indicates andecrease in autoimmunity.

[0061] In Vitro Assay for Monitoring Cell Death

[0062] The present invention provides a novel assay for monitoringapoptosis of autoimmune cells in a mammal. In one preferred embodiment,the present invention provides an assay involving (a) isolating a bloodsample from a mammal, preferably a human, and (b) testing the bloodsample in vitro for killing of autoimmune cells compared tonon-autoimmune cells using techniques available in the art. As mentionedabove, non-autoimmune cells are generally resistant to TNF-alpha inducedapoptosis. An increase in cell death in autoimmune cells compared tonon-autoimmune cells indicates that the dose of TNF-alpha or other celldeath inducing agent is sufficient to induce killing of the autoimmunecells or defective bone marrow origin cells.

[0063] Combined TNF Induction Therapy

[0064] The present invention also features a drug combination thatincludes two or more TNF-alpha inducing agents. One particularlypreferred combined TNF-alpha treatment is the combination of TNF-alphaand IL1. This treatment strategy goes against the current dogmasurrounding treatment of autoimmune disease. For example, at the TNFSecond International Meeting (A Validated Target with MultipleTherapeutic Potential, 24-25 February, 1999, Princeton, N.J., USA) itwas disclosed that a combination of anti-TNF-alpha antibody and anti-IL1would be advantageous in the treatment of autoimmune disease. Thetreatment of the present invention discloses induction of inflammation,which is the opposite of the treatment believed to be effective by thoseskilled in the art, that is, suppression of inflammation. Of course, inthe current treatment, inflammation does not occur because theinflammatory cells actually die prior to arriving at the target site orare killed at the target site.

[0065] Of course, the present invention is not limited to a combined TNFinducing therapy that includes only the combination of TNF-alpha andIL1, but includes any combination of TNF-alpha-including therapies,e.g., vaccination with BCG etc., viral infection, LPS, activation ofcells that normally produce TNF-alpha (i.e., macrophages, B cells, and Tcells), the chemotactic peptide fMet-Leu-Phe, bacterial and viralproteins that activate NF_(κ)B, agents that induce signaling pathwaysinvolved in adaptive immune responses (i.e., antigen receptors on B andT cells, CD28 on T cells, CD40 on B cells), agents that stimulatespecific autoreactive cell death receptors (i.e., TNF, Fas (CD95), CD40,p75NF, and lymphotoxin Beta-receptor (LtbetaR), drugs that stimulateTNF-alpha converting enzyme (TACE) which cleaves the TNF-alpha precursor(i.e., to provide biological activity capable of stimulating enhancedproduction or enhanced cytokine life after secretion) etc.

[0066] Identification of Inflammation-Inducing Agents

[0067] In preferred embodiments, the present invention providesinflammatory agents for the treatment of autoimmune disease that arecounter to the anti-inflammatories used to treat autoimmune diseasestoday. For example, current methods for treating autoimmune diseaseinclude TNF-alpha antagonists. Thus, the present invention providesTNF-alpha agonists (i.e., chemicals, peptides, or antibodies) that acton a TNF-alpha receptor. Other preferred treatments could fall under thecategories of drugs that act in opposite to anti-TNF-alpha agonists,anti-TNF-alpha antibodies, TNFR2 fusion proteins (Immunex), Embrel,anti-IL1 therapies, TNF-alpha convertase inhibitors, p38 MAP kinaseinhibitors, phoshodiesterase inhibitors, thalidomide analogs, andadenosine receptor agonists.

[0068] In another preferred embodiment, the invention allows for theidentification of drugs that induce cell death or selectively hamper theautoimmune cells by binding to cell surface receptors or interactingwith intracellular proteins. For example, drugs that stimulate the IL-1pathway or drugs that interact with converging pathways such as Fas,FasL, TACI, ATAR, RANK, DR5, DR4, DCR2, DCR1, DR3, etc. The drugs of thepresent invention can be characterized in that they only kill autoimmunecells having a selective defect in a cell death pathway which can becharacterized by two distinct phenotypes, (1) defects in antigenpresentation for T cell education and (2) susceptibility to apoptosis.

[0069] It will be appreciated that the above-described assay formonitoring death of autoimmune cells can be used to identify novelTNF-alpha inducing agents and other inflammatory agents useful in thepresent invention. In preferred embodiments, autoimmune cells (i.e., anautoimmune cell isolated from a mammal diagnosed with autoimmunedisease) are exposed to a putative inflammatory or TNF-alpha inducingagent and assayed for increased cell death, an increase in cell death ofautoimmune cells compared to non-immune cells indicating identificationof a drug according to the present invention. Furthermore, autoimmuneblood could be exposed to chemical libraries for preferred and selectivecell death of yet unknown targets compared to non-autoimmune cells. Awide variety of chemical libraries are available in the art and can bescreened by use of the assay of the invention, which measures the rateof apoptosis of autoimmune cells.

[0070] In a related aspect, the above-described assay for monitoringdeath of autoimmune cells can be used to identify autoimmune cellshaving the two distinct phenotypes described above. In contrast totypical genetic approaches for identifying cells carrying geneticdefects, sensitivity to cell death may serve as the initialidentification marker. Once cell-death sensitive cells are identified,they can be assessed as to whether they also have the class I antigenpresentation defect. Thus, the present invention provides a method ofidentifying autoimmune cells by (1) assaying the cells for asusceptibility to apoptosis and (2) assaying for defects in antigenpresentation required for T cell education.

[0071] A Novel Combination Therapy

[0072] The data presented in Table 1 and described in detail in Example1, below, demonstrate the remarkable success of combining two methods toinduce long-term normoglycemia with islet allograft transplantation inan already diabetic NOD host. The invention combines two therapies aimedat two separate targets of the immune system. The invention tests thisconcept by combining my prior transplantation technology with anautoimmune strategy to thwart the underlying disease, and for the firsttime provides long-term normoglycemia in naturally diabetic hosts viatransplantation with allogeneic islets. Thus, the invention, views therejection problem as one involving two immune barriers, i.e., the graftrejection barrier and the recurrent autoimmunity barrier. To address thegraft rejection barrier, I used donor antigen modified islets, and forthe recurrent autoimmune barrier I used CFA, a strong inducer ofTNF-alpha. TABLE 1 Donor Host Individual Survival (days) Groups StrainStrain Treatment Days of normoglycemia Mean 1. C57BL/6 NOD-IDDM* — 2, 2,3, 9, 23 7.8 2. β₂M^(−/−) NOD-IDDM — 5, 9, 12, 12, 17, 18, 71 20. 3.C57BL/6 BALB/C — 5, 5, 7, 10 6.7 4. β₂M^(−/−) BALB/C— >100, >100, >100, >100 >100 5. NOD NOD-IDDM — 5, 10, 12, 13 10 6. NODNOD-IDDM CFA 12, 26, 30, >38, >66, >59 >120, >122 7. C57BL/6 NOD-IDDMCFA 10, 10, 10 10 8. β₂M^(−/−) NOD-IDDM CFA 5, 14, 32,36, >59, >79, >57 >115, >115

[0073] Table 1, above, represents a series of experiments that werecarried out in which host mice were treated to prevent recurrentautoimmunity, via killing or inactivation of autoreactive lymphocytes,and then transplanted with donor islet cells in which rejectiontriggering antigens had been eliminated or modified.

[0074] The mice were injected once intraperitoneally with completeFreund's adjuvant (CFA) (25 μl/mouse) to induce TNF-alpha.

[0075] The same day, islet cell transplantation was carried out asfollows. The donor antigen modified islet cells were isolated fromtransgenic β₂M (β2 microglobulin) knockout mice purchased from theJackson Labs. As mentioned above, the β₂M gene encodes a criticalchaperone protein essential for surface expression of class I proteins.Host β2M, a highly conserved protein, can in part re-constitute β₂M.

[0076] Transgenic or normal islet cells were transplanted into ninegroups of mice. Three of the groups (groups 6, 7, and 8) werepre-treated with CFA; the other six groups were not pre-treated.

[0077] As is shown in Table 1, naturally diabetic mice (NOD-IDDM) thatreceived transgenic transplants but were not pre-treated with CFA (group2) had mean survival times of 20 days, suggesting that the protection ofdonor tissue from graft rejection does not protect the tissue from anestablished autoimmunity. Likewise, group 7 establishes that hosttreatment with CFA, an immunomodulator now believed to modifyexclusively the autoimmune response, does not protect normal allogeneicdonor cells from rapid graft rejection. In contrast, the CFA treateddiabetic mice receiving transgenic transplants (groups 6 and 8) survivedover 57 days (mean). The remaining groups were additional controls:group 1 (no CFA; diabetic host, non-transgenic donor cells); group 3 (noCFA; non-diabetic host, non-transgenic donor cells); group 4 (no CFA;non-diabetic host; non-transgenic donor cells); and group 7 (CFA;diabetic host; non-transgenic donor strain). As is shown in Table 1, theonly one of these control groups exhibiting longevity were non-diabetichosts receiving transgenic donor cells, a therapy known to thwart graftrejection (group 4).

[0078] At approximately 120-130 days post transplantation, thetransplanted syngeneic and allogeneic islets were removed bynephrectomy. This was a control experiment to prove the animals revertedback to hyperglycemia.

[0079] The NOD mice receiving the syngeneic transplant had, within 24hrs, blood sugars in excess of 500 mg/dl and needed to be sacrificedimmediately because of their severe diabetic state. Histology on thesemice showed that the transplanted islets in the kidney survived in somecases but did not appear, in all cases, healthy. There were granulatedislets, but massive lymphocytic infiltrates surrounded and invaded theislet tissue. The islet invasion by host lymphocytes is a histologictrait indicative of autoreactivity against the islet tissue. Theendogenous pancreas demonstrated no surviving islets and was dotted withlarge lymphocytic clusters, presumably at sites of former islet tissue.

[0080] The NOD mice receiving the allogeneic islets, in contrast,remained normoglycemic after the nephrectomies had been performed toremove the allogeneic islet tissue. No change in blood sugar was noted.After approximately seven days of this perfect blood sugar control, themice were sacrificed. Histologic examination showed that endogenousislets in the pancreas were regenerated. The islet number was less thannormal, but the islets present were large, healthy, and had nolymphocyte invasion (although they did have a characteristic NOD rim oflymphocytes surrounding the healthy islet). In contrast, the allogeneicgrafts were gone in most cases by this late 120-140 daypost-transplantation time point. These results support the thesis thatwhat occurred was rescue and regeneration of the endogenous pancreas.The results support that the immune system was additionally re-educated.

[0081] Thus, in one preferred embodiment, the invention provides amethod of inhibiting rejection of transplanted islet cells in a diabeticpatient, by (a) pre-treating the islet cells to modify, eliminate, ormask islet cell antigen otherwise capable of causingT-lymphocyte-mediated rejection response in a patient, together with (b)treating the patient (prior to, during, or following transplantation) tokill or inactivate autoreactive host lymphocytes that are otherwisecapable of killing or damaging the transplanted islet cell.

[0082] In preferred embodiments, step (a) involves genetically alteringthe donor animal so that HLA class I or a molecule in its pathway isgenetically deleted or chaperone ablated to prevent surface expression,or masking HLA class I antigen using an antibody F (ab′)₂ fragment thatforms a complex with HLA class 1; and step (b) involves administering toa patient TNF-alpha, or a TNF-alpha inducing substance, e.g., tissueplasminogen activator (TPA), LPS, IL-1, TV light, such as aninteracellular mediator of the TNF-alpha signaling pathway or an inducerof cell death in defective cells.

[0083] Class I Antigen Presentation

[0084] Prior to the experiments described above, I observed that a smallamount of class I ablation was beneficial for the inhibition ofrejection of donor islets in diabetic NOD mice (Faustman et al., Science(1991) 252(5013):1700-2). Based on these results, I proposed that a morecomplete and permanent class I ablation might even be better for longterm graft survival. To achieve a more permanent class I ablation, Itransplanted F2 islets that were ablated for both the β₂M (β2microglobulin) gene and Tap 1 into already diabetic NOD mice (MHC classI^(−/−, −−)) (see Example 3). The β₂M gene encodes a critical chaperoneprotein essential for surface expression of class I peptides. The Tap 1gene encodes a protein required for transport of endogenousself-peptides into the endoplasmic reticulum for stable peptide andclass I assembly before presentation on the cell surface. Surprisingly,only one of the six mice exhibited long term graft survival. Individualgraft survival times (days) for the six mice were: 11, 12, 13, 14, 14,and 71. These unexpected results suggested that the reexpression ofpeptide and class I was a step that was not only not harmful, but wasactually necessary for immune system re-education leading to endogenousislet regeneration and rescue. Thus, in the present application, Ipropose, without limiting the biochemical mechanism of the invention,that some intact MHC class I molecules are required for the re-educationprocess to occur.

[0085] Based on the above-described results, it appears that the graftrejection barrier actually serves two important functions that appear tocontribute to successful islet cell regeneration in this model.Temporary class I ablation (class I^(−/−)) serves initially to protectthe graft from immediate rejection. Subsequently, MHC class I proteinsare reexpressed and exchanged on the graft by 24-72 hourspost-transplantation through abundant host β₂M proteins from the serum(Anderson et al., J. Immunol. (1975) 114(3):997-1000; Hyafil et al.,Proc. Natl. Acad. Sci., USA (1979) 76(11):5834-8; Schmidt et al.,Immunogenetics (1981) 13:483-91; Bernabeu et al., Nature (1984)308(5960):642-5; Li et al., Transplantation (1993) 55(4):940-6).Surprisingly, subsequent reexpression of endogenous peptide via MHCclass I appears to contribute to the reeducation of T lymphocytes withproper negative selection of autoreactive cells. In further support ofthis observation, I demonstrated, that to be therapeutically effective,at least one MHC class I K or D locus of the donor lymphocytes needed tobe matched to the corresponding locus in the NOD host mouse (i.e.,either the K^(d) or D^(b) locus should be present on the donor MHC classI expressing cells). Therefore, the reeducation component of thecombination therapy requires that the MHC class I expressing cells,presenting the self-peptide, be semi-syngeneic or fully syngeneic to theautoreactive cells in the NOD host mouse. Coupled with the selectiveelimination of autoreactive lymphoid cells by treatment with CFA, thepresent combination therapy provides a powerful treatment for autoimmunedisease where regeneration of tissue is desired.

[0086] Maintenance of Transplanted Islet Cells is not Required forRegeneration of Endogenous Pancreas

[0087] The experiments described above further suggested to me thatmaintenance of the transplanted islets in vivo might not be necessaryfor endogenous pancreatic islet cell regeneration. In order to test thistheory, transgenic MHC class I^(−/−) islet grafts were transplanted intoalready diabetic NOD mice with TNF-alpha induction. Transgenic MHC classI^(−/−) islet grafts placed under the kidney of diabetic NOD mice werelater removed by nephrectomy at various times post-transplantation. Asdescribed in Example 2, all mice remained normoglycemic for at least 120days after nephrectomy and the pancreatic histology revealed beautifulendogenous pancreatic islet regeneration. In contrast, NOD mice thatreceived syngeneic islet transplants rapidly returned to hyperglycemiapost-nephrectomy.

[0088] As proposed above, these data support the theory that forendogenous regeneration of islets, or other regenerating tissue subjectto immune attack (e.g., hepatic cells), maintenance of the transplantedislet cells is not essential to endogenous pancreatic islet cellregeneration and rescue. Thus, the invention, in one respect, views theproblem of tissue regeneration and rescue in autoimmunity as oneinvolving two different barriers, (i.e., the recurrent autoimmunitybarrier and the re-education barrier). The required steps for tissueregeneration appear to be: (1) ablate the host autoimmune cells (e.g.,by killing or inactivation); and (2) re-educate the immune system withclass I and peptide to protect the regenerating pancreas.

[0089] Thus, the present invention provides a method of reestablishingsystemic tolerance and eliminating existing autoimmunity that promotesregeneration and rescue of cells and tissue.

[0090] Treatment by Injection of MHC Class I and Peptide

[0091] Based on the discovery that class I peptide presentation isrequired for re-educating the NOD host and the knowledge thatmaintenance of transplanted islet cells is not required for endogenousislet cell regeneration, I proposed that islet transplantation andisolation might not be necessary for in situ islet regeneration insetting of autoimmunity. Islet isolation and transplantation arelaborious procedures with associated supply and demand limitations inthe clinical setting. A procedure for increasing the number offunctional islet cells that does not require islet isolation andtransplantation would provide great benefit to the treatment ofdiabetes. This method of treatment could be extended to other autoimmunediseases where immune reeducation is desired (U.S. Pat. No. 5,538,854).

[0092] I proposed that mere injection of functional cells expressingclass I (class I⁺), or even MHC class I/peptide complex, into a mammal,with concurrent ablation of autoimmune cells, would be efficacious intreating a diverse array of autoimmune diseases. For example, normalpancreatic islets express MHC class I and have few associated passengerlymphocytes that express both MHC class I and class II molecules (thispreparation is referred to herein as B6 splenocytes). In the case ofdiabetes, a preparation of normal pancreatic islet cells may be injectedinto a patient to achieve exposure to class I antigen. Although donorcell survival may be short lived, repeated exposure might be sufficientto re-educate the host immune system with concurrent ablation ofautoimmune cells. In cases where donor cell preparation is tedious orpoor donor cell survival time is limiting the efficacy of the method,class I/peptide complex may be administered directly to the host.

[0093] In order to test this hypotheses, diabetic NOD mice wereinitiated on a 40 day regimen of one bolus injection of CFA totransiently induce TNF-alpha and biweekly exposure by intravenousinjection to B6 splenocytes (class I⁺) (Example 4). As predicted, theinjected splenocytes survived only transiently in the host due torejection. However, transient elimination of autoimmune cells (e.g., viaCFA-mediated TNF-alpha induction) combined with repeat exposure to B6MHC class I and peptide was sufficient for reversal of diabetes inapproximately 30% of diabetic NOD hosts. Partial protection was achievedin approximately 50% of the diabetic NOD hosts, but the percentage ofhost NOD mice remaining normoglycemic increased to 80% after allowingthe islets to regenerate for more than 120 days. Furthermore, since thedonor B6 splenocytes have a MHC class I phenotype of K^(b)D^(b) and thehost NOD mice have a MHC class I phenotype of K^(d)D^(b), I was able todetermine that the now normoglycemic host NOD mice had insulin secretingpancreatic cells of donor B6 origin, based on K^(b) antibody staining.In addition, to support this finding, I determined that only injectionof live, non-irradiated, splenocytes resulted in a contribution of thedonor MHC class I phenotype to the host insulin secreting cells.

[0094] Blood sugar levels were poorly controlled in mice receiving theinjection therapy described above. Fluctuations in blood sugar levelcould negatively influence benefit of the combined injection therapy. Inorder to control for this variant, additional groups of diabetic NODmice were similarly treated with TNF-alpha induction and B6 splenocyteinjection, but with simultaneous intraperitoneal implantation of B6islets encapsulated with alginate (referred to herein as a euglycemicclamp). A euglycemic clamp provides a membrane barrier system thatallows short term glycemic control of insulin exchange but preventsdirect cell-cell contact (e.g., for T cell education). After 40 days,the encapsulated islets were surgically removed and blood sugar levelsof the diabetic NOD mice were monitored for evidence of in situ pancreasregeneration. Remarkably, diabetic NOD mice that had received biweeklyB6 splenocyte immunizations and a single dose of TNF-alpha inductiontherapy remained normoglycemic for 40 days after clamp removal in 78% ofthe cases. Moreover, after the therapy was halted and autoimmunityeliminated, the continuous expansion of the endogenous pancreas wassufficient for sustained tolerance to self antigens. In contrast, incontrol experiments, where splenocytes permanently ablated for MHC classI proteins (MHC class I^(−/−, −/−)), poor in situ islet regeneration wasobserved (Table 3, group 4, FIG. 5). However, injection of splenocyteslacking MHC class II proteins (MHC class II^(−/−)) permitted in situislet regeneration, presumably due to continued expression of endogenouspeptide in the context of MHC class I (Table 3, group 5, FIG. 5).Therefore reestablishment of self tolerance and elimination ofautoreactivity was MHC class I dependent.

[0095] Therefore, I have identified and optimized a novel combinationtreatment for diabetes melitus. Thus, in yet another aspect, the presentinvention features a method of increasing and preserving the number offunctional cells of a predetermined type in a diabetic patient thatincludes the steps of (1) ablation of autoimmune cells, (2) exposure toMHC class I and peptide, and (3) maintenance of glucose control. Asmentioned above, exposure may occur, for example, either bytransplantation of functional MHC class I and peptide presenting cellsof a predetermined type, or preferably by repeated injection of suchcells. Alternatively, exposure to MHC class I and peptide may occur byinjection of class I/peptide complexes, peptide feeding of autologouscells etc.

[0096] In a particularly preferred embodiment, the present inventionprovides a method of increasing the number of functional cells of apredetermined type in a diabetic patient that includes the steps of (1)ablation of autoimmune cells (i.e., cells that are defective in celldeath), (2) exposure to MHC class I and peptide by repeated injection offunctional cells of a predetermined type, expressing peptide in thecontext of MHC class I (or MHC class I/peptide complex), and (3)maintenance of glucose control. In the case of diabetes, the functionalcells of a predetermined type include islet cells, for example, B6splenocytes. Maintenance of blood glucose levels may be achieve by anymeans known in the art, for example, insulin injection, or by use of aeuglycemic clamp. The diabetic patient can be any mammal, preferably ahuman patient.

[0097] Treatment of Autoimmune Disease

[0098] Based on the discoveries described herein, I have devised a noveltherapy for the correction of any established autoimmunity. Used incombination, exposure to self peptide in the context of MHC class I andkilling or inactivation of autoreactive lymphocyte permits theendogenous regenerative potential of mammalian tissue to be enacted. Inaddition, the present treatment enables preservation and rescue ofexisting tissue. The effect of this combination therapy is there-education of the immune system with the simultaneous reversal ofautoimmunity within the host.

[0099] With respect to diabetes treatment, I further hypothesize thatsuccessfully regenerated pancreatic B6 islet cells that hyper-expressMHC class I and peptide (e.g., determined by histological examination)maintain peripheral tolerance once sufficient islet growth has beenestablished. In vivo exposure to MHC class I and peptide expressingcells by transplantation or injection appears to initiate theeducational process for long-term and stable tolerance, beyond theperiod of treatment.

[0100] Several striking similarities exist between the NOD mouse andhuman diabetic patients, suggesting that this novel therapeutic approachcan be easily applied to treat human diabetic patients. For example,diabetic human splenocytes, like murine splenocytes, have defects inresistance to TNF-alpha induced apoptosis (Hayashi et al., supra). Inaddition, like NOD mice, human splenocytes have age related defects inMHC class I presentation of self peptides for proper T cell selection(Faustman et al., supra; Fu et al., J. Clin. Invest. (1993) 91:2301-7).Finally, it has been recognized for years that even after a severehyperglycemic episode, diabetic humans continue to produceautoantibodies to islet targets, indicating that the islet cells orislet precursor cells of the pancreas were not completely ablated. Thisindicates that humans diagnosed with diabetic autoimmunity may have highislet regenerative potential.

[0101] Thus in one aspect, the invention features a method of increasingthe number of functional cells of a predetermined type in an individualdiagnosed with an autoimmune disease, by (1) providing a sample offunctional cells expressing MHC class I and peptide, (b) exposing amammal to the MHC class I and peptide, and (c) prior to, after orconcurrently with step (b), treating the mammal to kill or inactivateautoimmune cells (i.e., cells defective in apoptosis) in the mammal.

[0102] Where the mammal is a diabetic human patient, it may be desirableto add a further step of maintaining normal levels of glucose prior to,after, or concurrently with step (b). As described above, maintenance ofnormal blood glucose levels in a patient with established diabetes mayimprove the efficacy of the inventive method.

[0103] As mentioned previously, re-education of the immune system withMHC class I and peptide can employ cells expressing endogenous peptidein the context of MHC class I or class I /peptide complexes alone. Anumber of such immune system re-education methods are known, e.g., asdescribed in U.S. Pat. No. 5,538,854, hereby incorporated by reference.

[0104] Similarly, a variety of well known methods can be used in thepresent invention to accomplish ablation of autoimmune cells. Onepreferred treatment is the administration of TNF-alpha, which isavailable from Genentech Corporation, South San Francisco, Calif.;Roche; Boehringer Ingelheim; Asahi Chemical Industry; and SigmaChemicals. The administration intraperitoneally of TNF-alpha to decreaserejection in diabetes-prone mice is described in Rabinovitch et al., J.Autoimmunity (1995) 8(3):357-366, hereby incorporated by reference.

[0105] Other host treatment methods can be used as well to ablateautoimmune cells, for example, administration of CFA, interleukin-1(IL-1), proteasome inhibitors, NF_(κ)B inhibitors, anti-inflammatorydrugs, tissue plasminogen activator (TPA), lipopolysaccharide, UV light,or an intracellular mediator of the TNF-alpha signaling pathway. Suchagents induce the apoptosis of autoreactive lymphocytes by interruptingthe pathway downstream from TNF-alpha receptor signaling. Other usefulagents are drugs that act downstream of TNF-alpha receptor binding.(Baldwin et al., Ann. Rev. Immunol.(1996) 12:141; Baltimore, Cell (1996)87:13).

[0106] In other aspects, the invention features a method of increasingthe number of functional cells of a predetermined type in an individualdiagnosed with an autoimmune disease, by (a) providing a sample of cellsof the predetermined type, (b) treating the cells to modify thepresentation of an antigen of the cells that is capable of causing an invivo T-lymphocyte-mediated rejection response, (c) introducing thetreated cells into the mammal, and (d) prior to, after, or concurrentlywith step (c), treating the mammal to kill or inactivate T-lymphocytesof the mammal. This method may be particularly useful for the treatmentof advanced-stage autoimmune disease, where complete destruction of aparticular cell type or tissue has been achieved.

[0107] In preferred embodiments, step (b) involves eliminating, reducingor masking the antigen. A number of methods can be used to modify,eliminate, or mask donor cell antigens; some of these are described inthe afore-mentioned Faustman U.S. Pat. No. 5,283,058. For example, step(b) may involve genetically altering the donor animal so that HLA classI or a molecule in its pathway is genetically deleted or chaperoneablated to prevent surface expression. Alternatively, step (b) mayinvolve masking HLA class I antigen using an antibody F (ab′)₂ fragmentthat forms a complex with HLA class I.

[0108] The therapeutic regimen of the present invention can be used notjust to inhibit rejection of regenerating cells, but also to treatautoimmune diseases in which endogenous cell or tissue regeneration isdesired, e.g., to allow myelin regeneration (or mere preservation of theremaining autoimmune target cells that are surviving) in multiplesclerosis or joint regeneration in rheumatoid arthritis.

[0109] Where the invention is used not just to protect regeneratingendogenous cells, e.g., islet cells, from autoimmune attack, but also toprotect transplanted cells and tissues, the methods described above canbe combined with other, known methods for inhibiting allograftrejection. Such methods include administration of anti-alpha CD3antibodies, anti-CD40L antibodies (CD40 Ligand, a co-receptor for T celltriggering, to prevent reduction of tolerance in the host), FK506,tacrolimus, sirolimus, alpha-CD25 induction, etc. and cyclosporin A. Asis discussed above, autoimmune insulin-dependent diabetes melitus (IDDM)lymphocytes are particularly sensitive to cell death via the TNF-alphapathway, and thus drugs that potentiate this pathway downstream ofreceptor binding can be employed. Examples of such potentiating drugsare targets of TRIP, NIK, Ikk, TRADD, JUN, NF_(κ)B, Traf2, andproteasome processing etc.

[0110] Even when the primary goal is regeneration or rescue ofendogenous cells rather than permanent allograft engraftment, it can beuseful to implant an allograft and promote its temporary survival, whilesimultaneously promoting re-education of the immune system so that theendogenous cells can regenerate; autoreactive lymphocytes aredetrimental to both the allograft and the regenerating cells, andtherefore killing or inactivation of those cells is doubly advantageous.

[0111] Thus, in the case of diabetes, for example, transplanted isletscan be temporarily protected from rejection by temporary encapsulationor by meticulous blood sugar control with exogenous insulin, while thehost is treated, as described above, to kill autoreactive lymphocytesand the immune system is re-educated by methods using class I andpeptide or class II and peptide. An additional advantage of usingallogeneic islet transplants during this phase is that normal isletsthat are temporarily protected might provide normal hormonal andsecretory capacities which will optimize in situ regeneration andrescue.

Other Embodiments

[0112] The skilled artisan will appreciate that the present inventioncan easily be applied to treat any of a variety of autoimmune disorders.Particularly, the present invention is particularly preferred for thetreatment autoimmunity where destruction of a particular cell type ortissue is ongoing. The present invention provides the advantage ofproviding relief to patients with even established cases ofautoimmunity, where tissue destruction is advanced or complete. Thepresent invention will now be demonstrated by the following non-limitingexamples.

EXAMPLES Example 1 Combination Therapy

[0113] To devise a clinically applicable protocol for the regenerationof islets in a diabetic host, two therapies were combined and tested inthe diabetic NOD mouse (a murine model for human type I diabetes).First, donor B6 islets were protected from graft rejection by temporaryclass I ablation (class^(−/−)) of the β₂M gene (Anderson et al., supra;Hyafil et al., supra; Schmidt et al., supra; Bernabeu et al., supra; Liet al., supra). The transgenic donor B6 islets were then removed fromthe donor mouse and transplanted into the host NOD mouse. Subsequently,a single foot pad injection of CFA was simultaneously administered; atreatment that sustains levels of TNF-alpha for days (Sadelain et al.,Diabetes, (1990) 39:583-589; McInerey et al., Diabetes, (1991)40:715-725; Lapchak et al., Clin. Immunol. Immunopathol. (1992)65(2):129-134).

[0114] The response of severely diabetic NOD female mice to treatmentsof donor B6 islets with or without transient MHC class I^(−/−)interruption and TNF-alpha induction are summarized in Table 2. TABLE 2Blood Sugar Control in Diabetic NOD Mice Receiving Islet TransplantsTNF-alpha* Group Donor Induction Days of normoglycemia Mean ± SD #1 NOD− 4, 6, 6, 8, 10   9 ± 2.3 #2 B6 − 2, 2, 3, 9, 9, 23   8 ± 8.0 #3B6-Class I^(−/−) − 5, 9, 12, 12, 17, 18, 71 22 ± 24 #4 NOD + 30, 55, 61,70, 72, 121, 136, 137 85 ± 40 #5 B6 + 9, 9, 9, 10, 11  10 ± 0.9 #6†B6-Class^(−/−) + 13, 14, 14, 15, 25, 32, 32, 32, 36, >62 ±54  >133, >133, >131, >129, >132 #7†† B6-Class I^(−/−),^(−/−) + 11, 12,13, 14, 14, >148 35 ± 55

[0115] All hosts were female diabetic NOD mice, typically greater than20 weeks of age, with sustained blood sugar levels in excess of 400mg/dl for at least 7 days with the administration of insulin of 0.5 U/kgto prevent death. This dose of insulin typically maintains blood sugarlevels of NOD mice diabetic in the normal range of 100-200 mg/dl. Eightto twelve hours prior to transplantation, insulin is stopped. All islettransplants are performed unilaterally under the kidney capsule tofacilitate post-transplant islet histology using standard techniques.

[0116] Typically, NOD islets isolated form 5-10 week old pre-diabeticfemale NOD mice are rapidly rejected when transplanted into severelydiabetic NOD mice (Table 2, group 1). Similarly, B6 islets transplantedunder the kidney capsule of diabetic NOD mice are also rapidly rejectedwith a mean survival time of 8±8.0 (Table 2, Group 2). As published inthe literature, although donor islets with MHC class I^(−/−) ablationsurvive indefinitely in non-autoimmune hosts (Faustman, 1991, supra),the transient MHC class I ablation only permits a three fold increase inislet survival in the challenging diabetic NOD host. All diabetic NODhosts eventually reject the B6 class I^(−/−) A donor islets; meansurvival is extended to 22±24 days (Table 2, group 3). As shown in Table2, group 4, although TNF-alpha induction facilitates syngeneic islettransplantation in NOD hosts, this autoimmune directed therapy hasminimal effect of B6 islet survival. B6 islets transplanted intodiabetic NOD mice with TNF-alpha induction are uniformly rejected by day10 post-transplantation in all diabetic NOD recipients (Table 2, group5).

[0117] As shown in FIG. 1, B6 islets isolated from young NOD mice andtransplanted into a diabetic NOD mouse with TNF-alpha inductiondemonstrate severe lymphocytic infiltrates under the kidney capsule atthe islet transplantation site (see also, FIG. 2). At the same time,blood sugar levels have increased to what they were prior totransplantation. In addition, the endogenous pancreas shows no intactislets; the remaining isle structures in the pancreas are obscured bydense pockets of infiltrating lymphocytes. Similarly, B6 isletstransplanted into an NOD mouse treated with TNF-alpha induction arerejected; the histology is virtually indistinguishable; massivelypocytic infiltrates under the kidney capsule at the transplant sitewith the endogenous pancreas showing islet structures obliterated withlymphocyte invasion (FIG. 1B).

[0118] Importantly, combining MHC class I^(−/−) islet transplantationwith TNF-alpha induction in NOD diabetic hosts was successful (Table 2,group 6). Continuous and sustained normoglycemia was observed in 5 ofthe 14 diabetic NOD hosts; normoglycemia continued beyond 129 days afterislet transplantation in the previously diabetic NOD mice receiving thecombined treatment. The mean survival time for normoglycemia exceeded62±54 days. Long-term normoglycemic NOD mice were sacrificed after atleast 129 days of post-transplantation monitoring to evaluate thesubrenal capsule islet transplantation site and the endogenous pancreas.

[0119] Surprisingly, all 5 long-term normoglycemic NOD mice receiving B6class I^(−/−) islets with TNF-alpha induction treatment histologicallydemonstrate no surviving islet grafts under the kidney capsule at 130days post-transplantation. The endogenous pancreas of these micedemonstrated significant islet regeneration (FIG. 3). Furthermore, theislets in the pancreas lacked lymphocyte invasion or, at most,occasionally demonstrated circumferential lymphocytes surrounding theregenerated islets. As the individual animal histology in Table 3summarizes, in situ pancreas regeneration was exclusively a trait ofdiabetic NOD mice treated with TNF-alpha in combination withtransplantation of donor islets having transient MHC class I^(−/−)interruption. These results demonstrate that the above combinationtherapy (administration of TNF-alpha and islet cells temporarily ablatedfor class I (class I^(−/−))) successfully eliminates existingautoimmunity in severely diabetic NOD mice and promotes regeneration ofthe endogenous pancreas.

[0120] In addition to the elimination of the existing autoimmunity,elimination of TNF-alpha sensitive cells in NOD mice by combinedtreatment of CFA and C57 lymphocytes or islets was also observed. As thescientific literature reports, incubation of spleen cells fromnon-autoimmune C57BL/6 mice treated with TNF-alpha (10-20 ng/ml) had noeffect on cell viability. In contrast, high numbers of splenocytes fromfemale NOD mice were killed by apoptosis after 12 hours of low doseTNF-alpha exposure. TNF-alpha induced apoptosis in NOD spleen cells frommice 7-15 weeks of age was confirmed using either trypan blue stainingor flow cytometry (FIG. 7). Importantly, splenocytes from formerlydiabetic NOD mice which received the therapeutic combined treatment ofCFA plus class I positive cells were found to have permanent eliminationof TNF-alpha sensitive lymphocyte subpopulations (FIG. 7). In vitrotreatment of splenocytes from successfully treated NOD mice withTNF-alpha resulted in little or no cell death beyond that seen in theabsence of TNF-alpha.

[0121] I have also examined the time course of the appearance and theelimination of TNF-alpha sensitive subpopulations. In pre-diabetic NODmice, which progress to diabetes, splenocytes were found to have highsensitivity to TNF-alpha induced apoptosis beginning after approximately7 weeks of age. The timing of the onset of this sensitivity wascorrelated with a lineage/tissue specific decrease in LMP2 protein.Indeed, splenocytes from LMP2^(−/−) mice have TNF-alpha sensitivity atall ages. Yet, only the combined treatment of CFA and MHC class I donorcells permanently eliminated these TNF-alpha autoreactive cells inautoimmune NOD mice.

[0122] To further prove that the elimination of the TNF-alpha sensitivesub-population of splenocytes is a key feature of the combined treatmentof CFA and MHC class I donor cells, I conducted an adoptive transferexperiment. This was accomplished by testing whether diabetes, fromspontaneously diabetic NOD mice, can be transferred to healthyirradiated (790R) syngeneic young male NOD mice. In these experiments, Iobserved that spleen cells from diabetic NOD mice adaptively transferreddiabetes into healthy mice, typically within 20-30 days after aninjection of 2×10⁷ splenocytes (FIG. 8). Importantly, when thelymphocytes from diabetic mice were treated in vitro with TNF-alpha at20 ng/ml for 24 hours prior to transfer, diabetes induction was notobserved during the 33 days of observation (FIG. 8). Accordingly,treatment of splenocytes with TNF-alpha in vitro has the ability toselectively kill existing disease-causing cells. Although other cellpopulations may participate in disease expression, their action is notsufficient to enable expression of diabetes in the absence of theTNF-alpha sensitive population.

[0123] Furthermore, in these adoptive transfer experiments, all male NODmice receiving untreated or TNF-alpha treated diabetic lymphocytes, ifstill normoglycemic, were killed after the onset of diabetes or at day35. Male NOD mice receiving the untreated diabetic splenocytes becamehyperglycemic during the observation period and had their pancreaticislets obliterated by dense infiltrates. All male NOD mice receivingTNF-alpha treated lymphocytes remained normoglycemic, but had mild tomoderate invasive insulitis. This indicated that, while diabetestransfer was delayed, diabetes most likely would occur with lengthenedobservation times. Therefore, TNF-alpha treatment of diabeticsplenocytes delays rapid diabetes transfer but does not permanentlyeliminate T cells with the latent potential to cause disease in theautoimmune prone host.

[0124] Indeed, this experimental result is complementary to thepancreatic histology observed in the NOD mice exhibiting diabetes thatwere treated with CFA alone and shown in FIG. 1. Active autoimmunitydefined by invasive insulitis is never eliminated but is reduced inmagnitude by CFA treatment alone. To eliminate autoimmunity permanently,other interventions, such as re-introduction of syngeneic MHC class Iexpressing cells, as described herein, are required.

[0125] In addition, NOD diabetic mice successfully treated with thecombination of CFA and MHC class I donor cells no longer have TNF-alphasensitive lymphocyte subpopulations, indicating that this population ofdisease-causing cells has been eliminated and continues to bepermanently eliminated (FIG. 7). Therefore, not only does the brief invivo therapy with CFA and cells bearing semi-matched MHC class I peptidecomplexes eliminate existing autoimmune cells but also the combinedtherapy established permanent elimination of these disease causingcells. Splenocytes from a successfully treated long-term normoglycemicNOD mice do not transfer diabetes after 100 days of observation and thehost pancreatic histology revealed no lymphocytic infiltrates.

Example 2 Maintenance of Transplanted Islet Cells is not Required forRegeneration of the Endogenous Pancreas

[0126] To confirm the ability to eliminate existing autoimmunity andregenerate the endogenous pancreas, additional diabetic NOD mice weretransplanted with MHC class I^(−/−) islets under the kidney capsule withTNF-alpha induction. At various times post-transplantation, in thepresence of sustained induced normoglycemia, islet grafts placed underthe kidney were removed by nephrectomy and survival surgery performed toevaluate whether maintenance of normal blood sugar levels was dependenton presence of the graft. FIG. 3 shows that all five severely diabeticmice that successfully received TNF-alpha induction and B6 MHC classI^(−/−) islet therapy remained normoglycemic at sacrifice times 3 to 60days after nephrectomy. In addition, the pancreatic histology in allfive hosts revealed a surprising number of pancreatic islets, with minornumbers of circumferential lymphocytes or no lymphocytes surrounding theregenerated and rescued islets. Evaluation of all islet transplant sitesunder the kidney demonstrated no surviving transplanted islets.

[0127] In marked contrast, all NOD mice receiving syngeneic NOD isletcells by transplantation, in conjunction with TNF-alpha induction,rapidly returned to hyperglycemia post-transplantation, demonstratingfailure of this transplant protocol using syngeneic NOD islets topromote endogenous pancreatic islet cell regeneration.

Example 3 Temporary Class I^(−/−) Ablation is Critical for SuccessfulCombination Therapy Treatment

[0128] In order to begin to dissect the mechanism of systemicreestablishment of tolerance sufficient for pancreatic islet re-growth,additional experiments were performed. In order to achieve a highersuccess rate of pancreatic islet regeneration with eliminatedautoimmunity, a more permanent MHC class I ablated islet was tested inthe combination therapy treatment. Islets from B6 donors with bothablated β₂M and Tap 1 genes (MHC class I^(−/−,−/−)), the obligatorychaperone and transport proteins for MHC assembly, respectively, weretransplanted into severely diabetic NOD mice with TNF-alpha induction. Ifound that although this approach is effective at prolongingnormoglycemia in murine hosts without autoimmunity, this treatmentfailed to prolong normoglycemia in the autoimmune, diabetic NOD host.More permanent donor MHC class I elimination with TNF-alpha induction inthe diabetic NOD host culminated in rapid islet graft rejection and poorability to achieve endogenous islet regeneration (Table 2, group 7).Apparently, some expression of donor MHC class I and self peptide isessential for NOD tolerance induction to self antigens, even if only fora limited time, on average 20 days (Table 2, group 3) before transplantrejection.

Example 4 Injection of Temporarily Ablated Islet Cells is Sufficient forInduction of Endogenous Pancreas Regeneration

[0129] Based on the data of Example 3, above, I proposed that class I+lymphocyte immunizations could be an efficacious therapy, even if thedonor cells only survived a short time in vivo post-injection. In orderto test this theory, nine diabetic NOD mice with severe hyperglycemiawere initiated on a 40 day regimen of one bolus injection of CFA totransiently induce TNF-alpha and biweekly exposures by intravenous (IV)injection of B6 splenocytes (9×10⁶ splenocytes IV). B6 splenocytes are alymphoid cell population with intact MHC class I and self peptidepresentation that survives only transiently in vivo due to rejection bythe host.

[0130] Additionally, four diabetic control NOD mice were maintained overthe same time period with only insulin treatment. All control NOD micewere monitored every other day for hyperglycemia and insulin wasadministered daily unless normoglycemia returned. After approximately 40days of treatment, all control NOD mice receiving only insulin weredead. Poor blood sugar level control, cachexia, and weight lossaccounted for the uniform mortality of all diabetic NOD hosts by day 20(FIG. 4A). Control mice treated only with insulin also had pancreatichistology demonstrating impressive lymphoid infiltrates obscuring anyrecognizable islet structure (FIG. 4C).

[0131] In marked contrast, nine severely diabetic NOD mice receivingrepeat exposures to B6 splenocytes plus TNF-alpha induction were alivein eight of nine cases and three of the NOD mice had returned tonormoglycemia by day 40. In addition, four diabetic NOD mice treatedwith repeat B6 splenocyte immunization and TNF-alpha induction, hadimproved islet histology by day 40 (FIG. 4D). Pancreatic islets werevisible and lymphoid infiltrates were significantly reducedcircumferentially as well as adjacently to the islet structures. Thispattern is characteristic of a histology pattern of protective, notdestructive, lymphocyte infiltrates (Gazda et al., Journal ofAutoimmunity (1997) 10(3):261-70; Dilts et al., Journal of Autoimmunity(1999) 12(4):229-32). Three diabetic NOD mice with TNF-alpha inductionand B6 splenocyte immunizations produced complete islet regeneration andinsulin independence. Histology on these three mice revealeddramatically reduced lymphocytic autoreactivity and increased isletabundance (FIG. 4D). Therefore, combined treatment with TNF-alphainduction and repeated exposure to peptide-bound B6 MHC class Ilymphocytes was sufficient to transiently obliterate autoreactive Tcells and reverse NOD diabetes for at least 40 days in approximately 30%of the hosts tested (FIG. 4). The therapy was partially protective inapproximately 50% of the NOD hosts (FIG. 4).

[0132] In order to eliminate poorly controlled blood sugar levels as afactor hampering more complete islet regeneration, additional groups ofdiabetic NOD mice were similarly treated, with TNF-alpha induction andB6 splenocyte injection, but with simultaneous implantation of aeuglycemic clamp intraperitoneally, for 40 days. A murine euglycemicclamp in these studies consisted of alginate encapsulated B6 islets. Thealginate capsule provides a membrane barrier system that allows shortterm glycemic control of insulin exchange but prevents direct cell-cellcontact (e.g., for T cell education). After 40 days, the euglycemic NODmice with the encapsulated islets underwent surgical removal of thealginate capsules and the blood sugar levels of the diabetic NOD micewere monitored for evidence of in situ pancreas regeneration.

[0133] Table 3, shows that after 40 days, both mice treated with theeuglycemic clamp in the absence of TNF-alpha induction (Table 3,group 1) and mice treated with the euglycemic clamp and TNF-alphainduction (Table 3, group 2), showed an absence of endogenous isletregeneration and rapidly returned to hyperglycemia after clamp removal.Results indicate that under conditions of excellent glucose control andTNF-alpha induction, apoptosis of existing autoreactive cells is inducedduring the early phases of acute diabetes, but neither the degenerativestate of the pancreas (as assayed by histology) nor the course ofpreexisting autoimmunity can be altered. The histology of NOD controltreatment groups consisted of severe lymphocytic elimination of theislets in the pancreas (Table 3, FIG. 4). TABLE 3 Impact of short-term(40 day) blood sugar control on endogenous islet regeneration indiabetic NOD mice after removal of euglycemic clamp # of normoglycemicSpleen cell TNF-alpha recipients Group donor induction* Total # ofrecipients % 1 — − 0/7  0 2 — + 0/6  0 3 B6 + 7/9 78 4 B6 classI^(−/−),^(−/−) + 2/6 33 5 C57 class II^(−/−),^(+/+) +  8/11 73

[0134] In marked contrast, diabetic NOD mice that had received biweeklyB6 splenocyte immunizations in combination with a single dose ofTNF-alpha induction therapy remained normoglycemic for 40 days afterclamp removal in 78% of the cases. A total of nine diabetic NOD micewere treated with this therapy and seven of the nine NOD mice hadpancreatic histology that demonstrated sustained and continuing isletregeneration days to weeks after euglycemic clamp removal (Table 3, FIG.5). In general, host islets had circumferential lymphocyticaccumulations and in some cases were aldehyde fuschin positive, (i.e.,had excess insulin, beyond the amount to maintain normoglycemia).

[0135] Therefore, islet regeneration was optimized in establisheddiabetic NOD mice by maintenance of blood glucose levels (using aeuglycemic clamp), ablation of autoreactive lymphocytes (by briefTNF-alpha induction), and repeated exposure to MHC class I and peptidepresenting cells, after a 40 day course of bi-weekly B6 splenocyteinjections. Furthermore, after the therapy was halted and autoimmunityeliminated, the immediate rescue and continuous expansion of theendogenous pancreas was sufficient for sustained tolerance to selfantigens. The mechanism of splenocyte re-education was defined asdependent upon the education complex of MHC class I and endogenouspeptides. As demonstrated in Table 3, injection of splenocytespermanently ablated for MHC class I proteins (MHC class I^(−/−, −/−))into diabetic NOD mice with euglycemic clamps led to poor in situ isletregeneration (Table 3, group 4, FIG. 5). Injection of splenocyteslacking MHC class II proteins (MHC class II^(−/−)) permitted in situislet regeneration presumably due to continued expression of endogenouspeptide in the context of MHC class I (Table 3, group 5, FIG. 5).Reestablishment of self tolerance and elimination of autoreactivity wasMHC class I dependent and MHC class II independent. The sustainedeffectiveness of this treatment is demonstrated in FIG. 5. Blood sugarmaintenance was observed beyond 20 days after the removal of theeuglycemic clamp.

Example 5 Regeneration of the Endogenous Pancreas

[0136] Generally, the percentage of CD3⁺ T cells in young NOD mice (<12weeks of age) is low, but after 30 weeks of age the percentage of CD3⁺ Tcells increases dramatically and exceeds that of control mice. In orderto evaluate the impact of successful pancreas regeneration and rescue onNOD lymphocyte selection, flow cytometric analysis was performed on CD3⁺T cells from treated NOD mice.

[0137] Splenic CD3⁺ T cell percentages were evaluated in 5 treated NODmice receiving various treatments; represented in FIG. 6 at 5 to 26 daysafter treatment had stopped. An untreated age-matched NOD female mousetreated with insulin had 56% of splenocytes staining with anti-CD3antibodies. The age-matched B6 female mouse had 27% positive splenocytes(FIG. 6), a trend previously reported (Miyazaki Clin. Exp. Immunol.85:60,622; Pontesilli Clin. Exp. Immunol. 97:70,84). Two mice weresuccessfully treated through either B6 or B6 class II^(−/−) splenocyteimmunizations, in conjunction with TNF-alpha induction, and displayedpancreas rescue and regeneration. Remarkably, both successfully treatedNOD mice had 40% of splenocytes staining with anti-CD3⁺ antibodies (FIG.6). In marked contrast, unsuccessfully treated age matched NOD mice(treated with only TNF-alpha induction therapy or TNF-alpha inductiontherapy in conjunction with B6- MHC class I^(−/−) splenocytes) had noalterations in the high number of splenic CD3⁺ cells (FIG. 6).Therefore, the impact of halted autoimmunity and re-establishment oftolerance was systemic and included markedly altered T cell selectionthat partially normalized numbers of CD3⁺ T lymphocytes in the spleen.These observations were further affirmed by my results which showed that(1) successful therapy results in a substantial reduction in naive Tcells expressing CD45RB^(high), CD62L, and CD95 proteins and (2) anincrease in the number of long-term memory cells, both indications ofthe normalization of T cell selection as a result of the combinationtherapy.

Example 6 Identification and Elimination of Autoimmune Cells

[0138] TNF-alpha sensitivity, in the NOD mouse, results from signalingdefects in the NF_(κ)B/proteasome activation pathway, thereby permittingthe TNF-alpha combination treatment disclosed herein to be successful.Accordingly, the methods disclosed herein are applicable to any numberof mammalian systems. For example, I have shown that TNF-alpha inducedapoptosis also occurs in humans. In my studies using fresh humanlymphocytes, from patients with diverse autoimmune diseases, use of thecombination of CFA and MHC class I donor cells resulted in TNF-alphainduced apoptosis of autoreactive lymphocytes. Lymphocytes from patientswith type I diabetes (n=80), multiple sclerosis (n=5), and rheumatoidarthritis (n=10), were treated in vitro with 10-20 ng/ml of TNF-alphafor 24 hours and then evaluated for cell viability. Control lymphocytesfrom normal human donors were also studied (n=80). In all cases, thecontrol cells were found to be protected from cell death. In contrast,all lymphocytes from autoimmune samples showed varying degrees of celldeath after TNF-alpha exposure. These results support theabove-described notion that humans with diverse autoimmune diseases,similar to the NOD mice, have intracellular defects in signalingpathways such as NF_(κ)B, preventing normal viability after exposure tothis cytokine, TNF-alpha. Additional data from this study of humans hasrevealed that the severity of the autoimmune cell death after TNF-alphatreatment relates to the rate of onset of the disease not to theduration of the disease. This means, for example, that early onset ofdiabetic autoimmunity in a four-year old child is associated with agreater fraction of lymphocytes dying after TNF-alpha treatment than isobserved, following the same treatment, in lymphocytes from atwenty-year old person with onset of autoimmunity in early adulthood.Accordingly, it is likely that more aggressive autoimmune diseases,e.g., ones that appear earlier during a patient's lifetime, show agreater fraction of autoimmune cells susceptible to the treatment forautoimmune disease reversal as is discussed herein.

Example 7 Treatment of Sjorgen's Disease, RA, and Lupus

[0139] As detailed above, the therapeutic methods disclosed herein arereadily applicable to any number of autoimmune diseases. For example,when a TNF-alpha treatment regimen combined with exposure to MHC classI-self peptide comlexes was applied to other forms of autoimmunedisease, such as murine models of Sjogren's disease, lupussymptomatology, and rheumatoid arthritis, I determined that autoimmuneinfiltrates indicative of Sjogren's disease were eliminated in thesalivary and lachrymal glands, autoantibodies indicative of lupus wereeliminated, and in a model of rheumatoid arthritis (RA), treatment offemale RA mice with the therapy of this application halted limbswelling. The endpoint of a therapy effectiveness was monitored by grossinspection, histology of the target organ, and serology. With respect toRA, after disease onset, nearly 90% of the mice with severe RA weresuccessfully treated and the accompanying joint disease was no longerdetectable by gross inspection or by histologic evidence at the time ofdeath.

Example 8 In Vitro Monitoring

[0140] Treatment Scenario II

[0141] Prescreening: A human subject presenting symptoms of type Idiabetes will be brought into the clinic to give a single blood donationthat will be divided into two tubes. One tube will be used to screen forthe presence of autoantibodies and the other tube will be used in an invitro screen for apoptotic cell death (e.g., TNF-induced) or acceleratedcell death due to any environmental or chemical agent. This initialsample will be used to obtain a base line C-peptide level and to verifythe absence of functional islets. Heightened in vitro sensitivity tocell death by any apoptotic cell death pathway will be a prerequisite tofuture therapy.

[0142] Treatment of Juvenile Onset Diabetes: If autoantibodies to isletcells are present, we will conclude that the pancreas is stillattempting to regenerate. Thus, if existing autoimmunity was eliminatedby treatment, the islets could successfully regrow.

[0143] An inexpensive approach to try to immediately rescue the pancreaswould be to repeatedly perform BCG administrations, as a non-specificimmunostimulant that could successfully raise the levels of endogenousTNF-alpha activity. Endogenous TNF-alpha will kill only the autoimmunecells (i.e., cells with a defect in protection from apoptosis).Initially we will start out with weekly BCG immunization. Blood sampleswill be collected within 24-48 hours after BCG immunization and testedin vitro (in cell culture) for the persistence of TNF-alpha sensitiveautoimmune cells. The isolated cells, grown in cell culture, will beexamined to determine whether the autoimmune cells, sensitive to death,are eliminated or reduced by this administration.

[0144] If the response to BCG immunization is positive, we will thenstart immunization with donor lymphocytes. Ideally, these lymphocyteimmunizations could be from both parents and would involve weeklyinfusions into the diabetic child, to be administered simultaneouslywith the BCG immunizations. In some cases, the donor lymphocytes will beirradiated to decrease the risk of infection transfer. We will introducethe lymphocytes intravenously at a dose of approximately 9×10⁶ to 9×10⁹as a start. With the in vitro monitoring assay, we expect to be able toidentify an optimal dose. Our goal is to continue this treatment from aminimum of 40 days up to about 6 months, or until positive human Cpeptide is found in the blood and insulin dosing is reduced. Early signsof possible tolerance induction success might not only be the presenceof C peptide but also the absence of TNF-alpha sensitive (autoreactive)cells. This would be an indication that the re-education is complete.Also, we predict that the phenotype of peripheral lymphoid cells wouldpossibly convert to a more mature phenotype (shown by an decreasedCD45RA to CD45RO ratio). Thus, screening in the lab will involve celldeath assays and lymphocyte surface markers of improved T cellselection. We expect also to see gross changes, such as reselection ofperipheral T cells and gross numbers of CD3 cells decreasing due toreintroduction of lymphoid cells directly, or due to regeneration ofislets that re-select peripheral T cells.

[0145] Treatment of Adult Onset Diabetes: If the patient is 40 years oldand has had diabetes for 20 or more years, we will follow the sametreatment protocol, but extend treatment over a longer period of time.In long standing disease, it is possible that the islet precursor cellsof the pancreas are effectively inactive and can no longer multiplybecause of years of autoimmune attack. This treatment might protect thepatient from complications of the disease, which in some cases may bedirectly related to the altered cytokines of the poorly selectedlymphoid cells causing fibrosis. In addition, this treatment mighteliminate the autoreactive cells that cause fibrosis and othercomplications. Lastly, this treatment might allow, for the first time,for islets to be transplanted with the barrier being only isletsurvival, not islet survival from graft rejection or islet survivalagainst autoimmunity.

[0146] Treatment Scenario II

[0147] Subjects. Patients who are older than age 18 but younger than 45years and who have Type 1 diabetes (insulin dependent and ketosis prone)will be recruited for this study. Participants will have to have aduration of Type 1 diabetes, dated from the time of insulinadministration, of at least one month, but not more than 5 years. Therationale for the duration criterion is predicated on the expectationthat persons with less than 5 years duration will still have residualbeta cell mass which is capable of recovery. Patients will be screenedto determine whether they have autoantibodies (anti-GAD and anti-IA-2 orislet cell antibodies) present. In addition, the presence of functioningbeta-cell mass, as measured by detectable (>0.2 pmol/ml) C-peptidelevels after glucagon stimulation, will be determined, although it willnot be a requirement for inclusion in the study. Exclusion criteria willinclude persons who have had previous BCG vaccination, a history ofclinical tuberculosis, or positive PPD test, a positive response to anintermediate (5 IU) PPD test, or any acute or chronic skin conditions.

[0148] Study Procedures: Eligible volunteers, as judged by chart review,will be asked to come to the Diabetes Research Center where they willhave measured both fasting and stimulated (6 min after 1 mg glucagongiven IV) C-peptide (endogenous insulin) secretion. In addition, bloodsamples will be obtained to measure autoantibody status (see above) andthe level of TNF and autoreactive T-lymphocytes or peripheral lymphoidcells with apoptosis sensitivity. Finally, if no recent (within fourweeks) hemoglobin A1c level is available, one will be obtained. Astandard panel of liver function tests and a CBC with differential willalso be obtained.

[0149] TNF-alpha induction, for example, by CFA administration or BCG“vaccination,” will be performed with a standard method with apercutaneous injection of 0.3 ml of a 50 mg/ml solution(Organon)(equivalent to a TNF-alpha induction of 10 μg/m²) into thedeltoid area. After the BCG solution is topically applied to the skin, asterile multipuncture disc is used to administer to BCG percutaneously.

[0150] Volunteers will be asked to return at weekly intervals for fourweeks to have a blood specimen obtained to repeat measurements of TNFand auto-reactive lymphocytes (1 green top and 1 red top tube). Thevaccination site (deltoid area) will be examined on each of theseoccasions to determine whether any significant ulceration or localreaction has occurred. In addition, patients will be questioned withregard to any febrile or other systemic symptoms that may have occurred.After four weeks, the patients will have a repeat vaccination performedin the opposite arm and similar, weekly monitoring will go on foranother two months.

[0151] Depending on the results from the first group of 5 subjects,adjustments in dosage and/or frequency of BCG vaccination will be madefor a subsequent group of 5 individuals.

[0152] Risks: The risks entailed in this study are minor and include theminor discomfort of obtaining blood samples. The total volume of bloodobtained over the course of the three-month study will be considerablyless than usually given in a single blood donation. The glucagonstimulated C-peptide test is commonly used in experimental protocols.The glucagon injection may be associated with mild nausea which usuallydissipates in 5 minutes. Rarely (less than 1 in 20) subjects may vomitafter glucagon.

[0153] BCG vaccination have been used for more than 30 years in manycountries, including Canada and in western Europe, as a vaccinationagainst tuberculosis. The recognized side effects of BCG vaccinationinclude mild local discomfort at the vaccination site with a papularrash developing at the site 10-14 days after vaccination and reaching amaximal diameter of 3 mm 4-6 weeks after vaccination. The rash may scalethereafter, and rarely leaves a visible scar. Local adenopathy is rarelyseen in children, but almost never in adults. Rare events includeosteomyelitis, lupoid reactions, disseminated BCG infections and death.The frequency of these severe reactions is between 1 in 1,000,000 and 1in 5,000,000 vaccinations, and have occurred almost exclusively inimmunosuppressed children. Most of the recent experience with BCG hasbeen in the intravesicular treatment of bladder cancer, where weeklyinstallations of BCG are performed for ≧6 weeks. Finally, BCGvaccination has been used in Type 1 diabetes without any adverseconsequences noted.

[0154] All references cited herein are hereby incorporated by referencein their entirety.

What is claimed is:
 1. A method of increasing or maintaining the numberof functional cells of a predetermined type in a mammal, comprising thesteps of: a) exposing said mammal to MHC class I and peptide, and b)prior to, after, or concurrently with step a), treating said mammal tokill or inactivate autoimmune cells of said mammal.
 2. The method ofclaim 1 wherein step a) comprises exposing said mammal to a MHC classI/peptide complex or exposing said mammal to cells capable of expressingMHC class I and peptide.
 3. The method of claim 1, wherein said methodfurther comprises maintaining the blood glucose level in said mammalwithin a normal range.
 4. A method of increasing or maintaining thenumber of functional cells of a predetermined type in a mammal, saidmethod comprising the steps of: a) providing a sample of cells of saidpredetermined type, b) treating said cells to modify the presentation ofan antigen of said cells that is capable of causing an in vivoautoimmune cell-mediated rejection response, c) introducing said treatedcells into said mammal, and d) prior to, after, or concurrently withstep c), treating said mammal to kill or inactivate autoimmune cells ofsaid mammal.
 5. The method of claim 4, wherein said mammal is a humanpatient.
 6. The method of claim 5, wherein said cells areinsulin-producing islet cells.
 7. The method of claim 4, wherein step b)comprises eliminating, reducing, or masking said antigen.
 8. The methodof claim 4, wherein step d) comprises administering to said mammalTNF-alpha or a TNF-alpha inducing substance.
 9. The method of claim 8,wherein the TNF-alpha inducing substance is tissue plasminogenactivator, LPS, interleukin-1, UV light, or an intracellular mediator ofthe TNF-alpha signaling pathway.
 10. The method of claim 1, wherein saidmammal has a mutation in the lmp2 gene.
 11. The method of claim 4,wherein said mammal has a mutation in the lmp2 gene or equivalentthereof.
 12. A method of increasing the number of functional cells of apredetermined type in a mammal, said method comprising the steps of: a)treating said mammal with an agent that kills or inactivates autoimmunecells of said mammal; b) periodically monitoring the cell death rate ofsaid autoimmune cells; and c) periodically adjusting the dosage of saidagent administered to said mammal based on the monitoring of step b).13. The method of claim 12, wherein said agent comprises TNF-alpha, aTNF-alpha inducing substance, tissue plasminogen activator, LPS,interleukin-1, UV light, or an intracellular mediator of the TNF-alphasignaling pathway.
 14. The method of claim 8, wherein step d) comprisesadministering to said mammal two agents that increase TNF-alpha.
 15. Themethod of claim 12, wherein step a) comprises administering to saidmammal two agents that increase TNF-alpha.
 16. A method for diagnosingan autoimmune disease or the predisposition to said disease in a mammal,said method comprising the steps of: a) providing peripheral cells froma mammal, b) treating said cells with a TNF-alpha treatment regimen, andc) detecting cell death of said peripheral cells, wherein an increase incell death, when compared with control cells, is indicative of saidmammal having an autoimmune disease or a predisposition to said disease.17. The method of claim 16, wherein said peripheral cells comprisesplenocytes, T lymphocytes, B lymphocytes, or cells of bone marroworigin.
 18. The method of claim 16, wherein said mammal is a humanpatient.
 19. The method of claim 16, wherein said TNF-alpha treatmentregimen comprises treating said peripheral cell with TNF-alpha.